The advances in recent years in the field of lipoprotein system analysis have shown that the narrow correlations found in older epidemiological data between plasma cholesterol concentrations and the risk of early atherosclerosis, especially coronary sclerosis, derive essentially from high-cholesterol beta-lipoproteins. In human blood, about 70-80% of the total cholesterol is normally found attached to the low density or beta-lipoproteins, with the remainder being distributed in other lipoprotein fractions, essentially Very Low Density Lipoproteins, High Density Lipoproteins and chylomicrons. (As employed herein, VLDL means Very Low Density Lipoproteins; LDL means Low Density Lipoproteins; and HDL means High Density Lipoproteins.) In pathological processes accompanied by a disturbed fat metabolism or increased plasmalipid concentrations and leading to early atherosclerosis, the percentage of beta-lipoprotein or beta-cholesterol in the total cholesterol may even increase further. This means that hypercholesterolemia is caused, as a rule, by hyper-beta-lipoproteinemia. In the past, therefore, many attempts have been made (1) to measure the low density lipoproteins selectively, and (2) to eliminate them from the circulating blood, selectively if possible.
Neither objective has been achieved satisfactorily until now, taking into consideration the state of the art. The usual methods of quantifying low density lipoproteins are based either on the use of the division of the lipoprotein spectrum into density classes with the aid of an ultracentrifuge, or on the division of the lipoprotein spectrum in the electric field, a method utilized in lipoprotein electrophoresis. Also there are precipitation processes which are based on the fact that apo-B-containing lipoproteins (VLDL and LDL) can be separately determined by polyanions and divalent cations from non-apo-B-containing lipoproteins by precipitation of the former. Apo-B-protein is the main protein component of the low density or beta-lipoproteins as well as of the VLDL or pre-beta-lipoproteins and chylomicrons.
The precipitation techniques have hitherto not been suitable for separating VLDL from LDL. The two first-named methods (i.e., ultracentrifugation, for which, see R. J. Havel, H. A. Eder and J. H. Bragdon: The Distribution and Chemical Composition of Ultracentrifugally Separated Lipoproteins in Human Serum, J. Clin. Invest., 34, 1345 (1955); and electrophoresis, for which, see H. Wieland and D. Seidel: Fortschritte in der Analytik des Lipoproteinmusters-Advances in analyzing the lipoprotein sample, Inn. Med., 5, 290-300 (1978)) have the disadvantage in relatively large routine series that they were either too expensive and time-consuming or could not be automated. In addition, direct measurement of the low density cholesterol was possible only after isolation of the fractions, usually with the use of an ultracentrifuge. The mathematical determination of the low density cholesterol component by electrophoresis presupposes certain premises of the protein lipid composition. The same applies to the methods which utilize precipitation techniques. The conventional precipitation techniques also have the disadvantage that they divide the lipoprotein spectrum into only two main fractions (apo-B-containing and non-apo-B-containing), that is, a distinction between or separation of VLDL and LDL is not possible.
Therapeutic efforts of effectively reducing the low density lipoproteins have thus far all been unsatisfactory. Control of the low density lipoprotein concentration by medication is extremely difficult and as a rule unsatisfactory, especially in the genetic forms of fat metabolism disorders. In the results obtained theretofore by medication, the therapeutic success of lowering the atherosclerosis risk could not be proven with certainty. Surgery, based essentially on the application of abnormal circulatory conditions or on displacement of major portions of intestine, have indeed given clear therapeutic results but cannot be regarded as generally acceptable because of the anticipated strong side effects. Such drastic therapeutic manipulations have shown, however, that with a sufficient reduction of the low density lipoproteins, not only can atherosclerotic processes be stopped, but existing atherosclerotic vessel alterations are reversible.
"Mechanical" elimination of low density lipoproteins from the blood has been tried heretofore essentially in two ways. One involves treatment of the affected patients by a so-called plasmapheresis, a method involving complete exchange of the entire blood serum. While this method has had the result of lowering the low density lipoprotein content of the affected patients, at the same time those lipoproteins which counteract atherosclerosis (high density lipoproteins) are also eliminated from the blood, on balance an undesirable therapeutic effect. Moreover, in the plasma exchange all other proteins of the plasma, including coagulation factors, globulins and hormones, are eliminated as well. Yet this method has proved quite useful for certain cases of hyper-beta-lipoproteinemia, although the search for a selective elimination of the low density lipoproteins from the plasma continues to have a high priority.
A second method of eliminating low density lipoproteins from the blood was undertaken with the aid of specific antibodies which were coupled to a matrix. The antibodies were obtained by immunization of sheep or rabbits. Although a drastic lowering of all apo-B-containing lipoproteins was achieved by such method (see A. Habenicht, Inaugural Dissertation, 1978, Heidelberg, School of Med.; W. Stoffel and Th. Demant, Selective Removal of Apolipoprotein B-containing Serum Lipoproteins from Blood Plasma, Proc. Nat. Acad. Sci. U.S.A., 78, 611-615 (1981)), the method has the disadvantage that during therapeutic application the antibodies produced in an animal inevitably get into the circulation of the treated patient, albeit in small quantities. In the long run this is bound to cause immunological problems in the person to be treated. This considerable objection to such method is the more serious because the treatment of fat metabolism disorders must be lifelong if it is to be effective and enduring. It must be noted that, especially in hereditary disorders, the therapy must begin at a youthful age in order to stop or to retard substantially the process of premature atherosclerotic vascular disease. Another disadvantage of the use of immobilized antibodies to Apo-B is that of unspecific elimination of all apo-B-containing lipoproteins. This method, therefore, is not apt to selectively eliminate low density lipoproteins as the only atherogenic lipoproteins. A major disadvantage of the simultaneous removal of VLDL is the resulting stimulation of triglyceride and cholesterol synthesis in the liver.
A third method of removing low density lipoproteins from the blood is based on the selective absorption of beta- and pre-beta-lipoproteins from whole blood by sulfated polysaccharides coupled to agarose beads, e.g., heparin or dextran according to U.S. Pat. No. 4,103,685. A disadvantage evident from the description in the patent is the low capacity of this method for eliminating the lipoproteins, as well as the fact that not only low density but also very low density lipoproteins are removed. Cholesterol reductions are described according to the method of U.S. Pat. No. 4,103,685 only for in vitro tests. As is evident from the accompanying reduction of the phospholipids and triglycerides, the cholesterol reduction appears to have been caused by a dilution of the patient's blood rather than by selective elimination.
The procedure described in U.S. Pat. No. 4,215,993, of precipitating lipoproteins generally at their isoelectric point, relates, in contrast to the method of the present invention, to the use of sodium-phosphorus tungstate. This polyanion is unphysiological and therefore, in contrast to the present process, unsuitable for use as a therapeutic treating agent. Moreover, when employing phosphorus tungstate it is not possible to eliminate or to determine low density lipoproteins selectively, so that the main objective of the present invention is not achieved. That this is so was indicated in U.S. Pat. No. 4,215,993 by, among other things, the recommendation to calculate the low density lipoproteins according to a mathematical formula (Friedewald formula) from the so-called HDL cholesterol, taking into consideration also the total cholesterol and the triglycerides. Essentially the isoelectric point precipitation using phosphorus tungstic acid, as described in such patent, does not differ in its result from the usual polyanion precipitation using bivalent cations. A complete precipitation of VLDL and LDL in such process is achieved only when polymers, such as polyvinyl pyrolidone or polyethylene glycol, of exactly defined molecular weight are added to the precipitation solution. That this cannot be achieved in an extracorporeal system is beyond question.
It should also be pointed out that U.S. Pat. No. 4,215,993 misleadingly refers to the totality of beta-lipoproteins, pre-beta-lipoproteins and chylomicrons as low density lipoproteins. Since it is a declared object of the process described in such patent to determine high density lipoproteins in the serum, it is important that the VLDL, chylomicrons and LDL occurring along with high density lipoproteins be eliminated by this precipitation method. A specific precipitation of the real low density lipoproteins or beta-lipoproteins was neither intended nor achieved.
We have now discovered processes whereby low density lipoproteins or beta-lipoproteins can be removed selectively and at a high degree of completeness from blood, i.e., from whole serum or from blood components such as plasma. When heparin is employed, such process may be employed for therapeutic purposes as well as for diagnostic purposes. We have also invented apparatus for practicing the process for therapeutic purposes.